TgMORN1 Is a Key Organizer for the Basal Complex of Toxoplasma gondii

Toxoplasma gondii is a leading cause of congenital birth defects, as well as a cause for ocular and neurological diseases in humans. Its cytoskeleton is essential for parasite replication and invasion and contains many unique structures that are potential drug targets. Therefore, the biogenesis of the cytoskeletal structure of T. gondii is not only important for its pathogenesis, but also of interest to cell biology in general. Previously, we and others identified a new T. gondii cytoskeletal protein, TgMORN1, which is recruited to the basal complex at the very beginning of daughter formation. However, its function remained largely unknown. In this study, we generated a knock-out mutant of TgMORN1 (ΔTgMORN1) using a Cre-LoxP based approach. We found that the structure of the basal complex was grossly affected in ΔTgMORN1 parasites, which also displayed defects in cytokinesis. Moreover, ΔTgMORN1 parasites showed significant growth impairment in vitro, and this translated into greatly attenuated virulence in mice. Therefore, our results demonstrate that TgMORN1 is required for maintaining the structural integrity of the parasite posterior end, and provide direct evidence that cytoskeleton integrity is essential for parasite virulence and pathogenesis.     

The Role of Clathrin in Post-Golgi Trafficking in Toxoplasma gondii

Apicomplexan parasites are single eukaryotic cells with a highly polarised secretory system that contains unique secretory organelles (micronemes and rhoptries) that are required for host cell invasion. In contrast, the role of the endosomal system is poorly understood in these parasites. With many typical endocytic factors missing, we speculated that endocytosis depends exclusively on a clathrin-mediated mechanism. Intriguingly, in Toxoplasma gondii we were only able to observe the endogenous clathrin heavy chain 1 (CHC1) at the Golgi, but not at the parasite surface. For the functional characterisation of Toxoplasma gondii CHC1 we generated parasite mutants conditionally expressing the dominant negative clathrin Hub fragment and demonstrate that CHC1 is essential for vesicle formation at the trans-Golgi network. Consequently, the functional ablation of CHC1 results in Golgi aberrations, a block in the biogenesis of the unique secretory microneme and rhoptry organelles, and of the pellicle. However, we found no morphological evidence for clathrin mediating endocytosis in these parasites and speculate that they remodelled their vesicular trafficking system to adapt to an intracellular lifestyle.     

Computational Analysis and Experimental Validation of Gene Predictions in Toxoplasma gondii

Background

Toxoplasma gondii is an obligate intracellular protozoan that infects 20 to 90% of the population. It can cause both acute and chronic infections, many of which are asymptomatic, and, in immunocompromized hosts, can cause fatal infection due to reactivation from an asymptomatic chronic infection. An essential step towards understanding molecular mechanisms controlling transitions between the various life stages and identifying candidate drug targets is to accurately characterize the T. gondii proteome.

Methodology/Principal Findings

We have explored the proteome of T. gondii tachyzoites with high throughput proteomics experiments and by comparison to publicly available cDNA sequence data. Mass spectrometry analysis validated 2,477 gene coding regions with 6,438 possible alternative gene predictions; approximately one third of the T. gondii proteome. The proteomics survey identified 609 proteins that are unique to Toxoplasma as compared to any known species including other Apicomplexan. Computational analysis identified 787 cases of possible gene duplication events and located at least 6,089 gene coding regions. Commonly used gene prediction algorithms produce very disparate sets of protein sequences, with pairwise overlaps ranging from 1.4% to 12%. Through this experimental and computational exercise we benchmarked gene prediction methods and observed false negative rates of 31 to 43%.

Conclusions/Significance

This study not only provides the largest proteomics exploration of the T. gondii proteome, but illustrates how high throughput proteomics experiments can elucidate correct gene structures in genomes.     

The Inner Membrane Complex Sub-compartment Proteins Critical for Replication of the Apicomplexan Parasite Toxoplasma gondii Adopt a Pleckstrin Homology Fold

Toxoplasma gondii, an apicomplexan parasite prevalent in developed nations, infects up to one-third of the human population. The success of this parasite depends on several unique structures including an inner membrane complex (IMC) that lines the interior of the plasma membrane and contains proteins important for gliding motility and replication. Of these proteins, the IMC sub-compartment proteins (ISPs) have recently been shown to play a role in asexual T. gondii daughter cell formation, yet the mechanism is unknown. Complicating mechanistic characterization of the ISPs is a lack of sequence identity with proteins of known structure or function. In support of elucidating the function of ISPs, we first determined the crystal structures of representative members TgISP1 and TgISP3 to a resolution of 2.10 and 2.32 Å, respectively. Structural analysis revealed that both ISPs adopt a pleckstrin homology fold often associated with phospholipid binding or protein-protein interactions. Substitution of basic for hydrophobic residues in the region that overlays with phospholipid binding in related pleckstrin homology domains, however, suggests that ISPs do not retain phospholipid binding activity. Consistent with this observation, biochemical assays revealed no phospholipid binding activity. Interestingly, mapping of conserved surface residues combined with crystal packing analysis indicates that TgISPs have functionally repurposed the phospholipid-binding site likely to coordinate protein partners. Recruitment of larger protein complexes may also be aided through avidity-enhanced interactions resulting from multimerization of the ISPs. Overall, we propose a model where TgISPs recruit protein partners to the IMC to ensure correct progression of daughter cell formation.     

Neurons are the Primary Target Cell for the Brain-Tropic Intracellular Parasite Toxoplasma gondii

Toxoplasma gondii, a common brain-tropic parasite, is capable of infecting most nucleated cells, including astrocytes and neurons, in vitro. Yet, in vivo, Toxoplasma is primarily found in neurons. In vitro data showing that interferon-γ-stimulated astrocytes, but not neurons, clear intracellular parasites suggest that neurons alone are persistently infected in vivo because they lack the ability to clear intracellular parasites. Here we test this theory by using a novel Toxoplasma-mouse model capable of marking and tracking host cells that directly interact with parasites, even if the interaction is transient. Remarkably, we find that Toxoplasma shows a strong predilection for interacting with neurons throughout CNS infection. This predilection remains in the setting of IFN-γ depletion; infection with parasites resistant to the major mechanism by which murine astrocytes clear parasites; or when directly injecting parasites into the brain. These findings, in combination with prior work, strongly suggest that neurons are not incidentally infected, but rather they are Toxoplasma’s primary in vivo target.     

Dynamic Imaging of CD8+ T Cells and Dendritic Cells during Infection with Toxoplasma gondii

To better understand the initiation of CD8⁺ T cell responses during infection, the primary response to the intracellular parasite Toxoplasma gondii was characterized using 2-photon microscopy combined with an experimental system that allowed visualization of dendritic cells (DCs) and parasite specific CD8⁺ T cells. Infection with T. gondii induced localization of both these populations to the sub-capsular/interfollicular region of the draining lymph node and DCs were required for the expansion of the T cells. Consistent with current models, in the presence of cognate antigen, the average velocity of CD8⁺ T cells decreased. Unexpectedly, infection also resulted in modulation of the behavior of non-parasite specific T cells. This TCR-independent process correlated with the re-modeling of the lymph node micro-architecture and changes in expression of CCL21 and CCL3. Infection also resulted in sustained interactions between the DCs and CD8⁺ T cells that were visualized only in the presence of cognate antigen and were limited to an early phase in the response. Infected DCs were rare within the lymph node during this time frame; however, DCs presenting the cognate antigen were detected. Together, these data provide novel insights into the earliest interaction between DCs and CD8⁺ T cells and suggest that cross presentation by bystander DCs rather than infected DCs is an important route of antigen presentation during toxoplasmosis.     

Western Australian Marsupials Are Multiply Infected with Genetically Diverse Strains of Toxoplasma gondii

Five different organs from 16 asymptomatic free-ranging marsupial macropods (Macropus rufus, M. fuliginosus, and M. robustus) from inland Western Australia were tested for infection with Toxoplasma gondii by multi-locus PCR-DNA sequencing. All macropods were infected with T. gondii, and 13 had parasite DNA in at least 2 organs. In total, 45 distinct T. gondii genotypes were detected. Fourteen of the 16 macropods were multiply infected with genetically distinct T. gondii genotypes that often partitioned between different organs. The presence of multiple T. gondii infections in macropods suggests that native mammals have the potential to promote regular cycles of sexual reproduction in the definitive felid host in this environment.     

Global Metabolomic Profiling of Mice Brains following Experimental Infection with the Cyst-Forming Toxoplasma gondii

The interplay between the Apicomplexan parasite Toxoplasma gondii and its host has been largely studied. However, molecular changes at the metabolic level in the host central nervous system and pathogenesis-associated metabolites during brain infection are largely unexplored. We used a global metabolomics strategy to identify differentially regulated metabolites and affected metabolic pathways in BALB/c mice during infection with T. gondii Pru strain at 7, 14 and 21 days post-infection (DPI). The non-targeted Liquid Chromatography-Mass Spectrometry (LC-MS) metabolomics analysis detected approximately 2,755 retention time-exact mass pairs, of which more than 60 had significantly differential profiles at different stages of infection. These include amino acids, organic acids, carbohydrates, fatty acids, and vitamins. The biological significance of these metabolites is discussed. Principal Component Analysis and Orthogonal Partial Least Square-Discriminant Analysis showed the metabolites’ profile to change over time with the most significant changes occurring at 14 DPI. Correlated metabolic pathway imbalances were observed in carbohydrate metabolism, lipid metabolism, energetic metabolism and fatty acid oxidation. Eight metabolites correlated with the physical recovery from infection-caused illness were identified. These findings indicate that global metabolomics adopted in this study is a sensitive approach for detecting metabolic alterations in T. gondii-infected mice and generated a comparative metabolic profile of brain tissue distinguishing infected from non-infected host.     

Suggestive Evidence for Darwinian Selection against Asparagine-Linked Glycans of Plasmodium falciparum and Toxoplasma gondii

We are interested in asparagine-linked glycans (N-glycans) of Plasmodium falciparum and Toxoplasma gondii, because their N-glycan structures have been controversial and because we hypothesize that there might be selection against N-glycans in nucleus-encoded proteins that must pass through the endoplasmic reticulum (ER) prior to threading into the apicoplast. In support of our hypothesis, we observed the following. First, in protists with apicoplasts, there is extensive secondary loss of Alg enzymes that make lipid-linked precursors to N-glycans. Theileria makes no N-glycans, and Plasmodium makes a severely truncated N-glycan precursor composed of one or two GlcNAc residues. Second, secreted proteins of Toxoplasma, which uses its own 10-sugar precursor (Glc₃Man₅GlcNAc₂) and the host 14-sugar precursor (Glc₃Man₉GlcNAc₂) to make N-glycans, have very few sites for N glycosylation, and there is additional selection against N-glycan sites in its apicoplast-targeted proteins. Third, while the GlcNAc-binding Griffonia simplicifolia lectin II labels ER, rhoptries, and surface of plasmodia, there is no apicoplast labeling. Similarly, the antiretroviral lectin cyanovirin-N, which binds to N-glycans of Toxoplasma, labels ER and rhoptries, but there is no apicoplast labeling. We conclude that possible selection against N-glycans in protists with apicoplasts occurs by eliminating N-glycans (Theileria), reducing their length (Plasmodium), or reducing the number of N-glycan sites (Toxoplasma). In addition, occupation of N-glycan sites is markedly reduced in apicoplast proteins versus some secretory proteins in both Plasmodium and Toxoplasma.Animals, fungi, and plants synthesize Asn-linked glycans (N-glycans) by means of a lipid-linked precursor containing 14 sugars (dolichol-PP-Glc₃Man₉GlcNAc₂) (26). Recently we used bioinformatics and experimental methods to show that numerous protists are missing sets of glycosyltransferases (Alg1 to Alg14) and so make truncated N-glycan precursors containing 0 to 11 sugars (46). For example, Entamoeba histolytica, which causes dysentery, makes N-glycan precursors that contain seven sugars (Man₅GlcNAc₂) (33). Giardia lamblia, a cause of diarrhea, makes N-glycan precursors that contain just GlcNAc₂ (41). N-glycan precursors may be identified by metabolic labeling with radiolabeled mannose (Entamoeba) or glucosamine (Giardia) (46). Unprocessed N-glycans of each protist may be recognized by wheat germ agglutinin 1 (WGA-1) (GlcNAc₂ of Giardia) or by the antiretroviral lectin cyanovirin-N (Man₅GlcNAc₂ of Entamoeba) (2, 33, 41).N-glycans are transferred from lipid-linked precursors to sequons (Asn-Xaa-Ser or Asn-Xaa-Thr, where Xaa cannot be Pro) on nascent peptides by an oligosaccharyltransferase (OST) (28). For the most part, transfer of N-glycans by the OST is during translocation, although there are human and Trypanosoma OSTs that transfer N-glycans after translocation (34, 45).N-glycan-dependent quality control (QC) systems for protein folding and endoplasmic reticulum (ER)-associated degradation (ERAD), which are present in most eukaryotes, are missing from Giardia and a few other protists that make truncated N-glycans (5, 26, 53). There is positive Darwinian selection for sequons (sites of N-glycans) that contain Thr in secreted and membrane proteins of organisms that have N-glycan-dependent QC (12). This selection occurs for the most part by an increased probability that Asn and Thr will be present in sequons rather than elsewhere in secreted and membrane proteins. In contrast, there is no selection on sequons that contain Ser, and there is no selection on sequons in the secreted proteins of organisms that lack N-glycan-dependent QC.For numerous reasons, we are interested in the N-glycans of Plasmodium falciparum and Toxoplasma gondii, which cause severe malaria and disseminated infections, respectively.(i) There has been controversy for a long time as to whether Plasmodium makes N-glycans. While some investigators identified a 14-sugar Plasmodium N-glycan resembling that of the human host (29), others identified no N-glycans (6, 22).(ii) There is also controversy concerning whether the N-glycans of Toxoplasma, after removal of Glc by glucosidases in the ER lumen, contain either 7 sugars (Man₅GlcNAc₂), like Entamoeba (32, 33), or 11 sugars (Man₉GlcNAc₂), like the human host (16, 19, 26). If it is Man₅GlcNAc₂, then Toxoplasma uses the dolichol-PP-linked glycan predicted by its set of Alg enzymes (32, 46). If it is Man₉GlcNAc₂, then Toxoplasma uses the dolichol-PP-linked glycan of the host cell (16, 19, 26).(iii) Both Plasmodium and Toxoplasma are missing proteins involved in N-glycan-dependent QC of protein folding (5).(iv) We hypothesize that there may be negative selection against N-glycans in Plasmodium and Toxoplasma, because the N-glycans added in the ER lumen during translocation will likely interfere with threading of nucleus-encoded apicoplast proteins into a nonphotosynthetic, chloroplast-derived organelle called the apicoplast (21, 35, 37, 48, 52, 54). Nucleus-encoded apicoplast proteins have a bipartite signal at the N terminus, which targets proteins first to the lumen of the ER and second to lumen of the apicoplast. This bipartite signal has been used in transformed plasmodia where green fluorescent protein (GFP) is targeted to the apicoplast with the bipartite signal of the acyl carrier protein (ACP_leader-GFP), to the secretory system with the signal sequence only (ACP_signal-GFP), and to the cytosol with the organelle-targeting transit peptide only (ACP_transit-GFP) (55). Similar constructs have been used to characterize signals that target nucleus-encoded proteins of Toxoplasma to the apicoplast (11, 25).Here we use a combination of bioinformatic, biochemical, and morphological methods to characterize the N-glycans of Plasmodium and Toxoplasma and to test our hypothesis that there is negative selection against N-glycans in protists with apicoplasts.     

Rhoptry Proteins ROP5 and ROP18 Are Major Murine Virulence Factors in Genetically Divergent South American Strains of Toxoplasma gondii

Toxoplasma gondii has evolved a number of strategies to evade immune responses in its many hosts. Previous genetic mapping of crosses between clonal type 1, 2, and 3 strains of T. gondii, which are prevalent in Europe and North America, identified two rhoptry proteins, ROP5 and ROP18, that function together to block innate immune mechanisms activated by interferon gamma (IFNg) in murine hosts. However, the contribution of these and other virulence factors in more genetically divergent South American strains is unknown. Here we utilized a cross between the intermediately virulent North American type 2 ME49 strain and the highly virulent South American type 10 VAND strain to map the genetic basis for differences in virulence in the mouse. Quantitative trait locus (QTL) analysis of this new cross identified one peak that spanned the ROP5 locus on chromosome XII. CRISPR-Cas9 mediated deletion of all copies of ROP5 in the VAND strain rendered it avirulent and complementation confirmed that ROP5 is the major virulence factor accounting for differences between type 2 and type 10 strains. To extend these observations to other virulent South American strains representing distinct genetic populations, we knocked out ROP5 in type 8 TgCtBr5 and type 4 TgCtBr18 strains, resulting in complete loss of virulence in both backgrounds. Consistent with this, polymorphisms that show strong signatures of positive selection in ROP5 were shown to correspond to regions known to interface with host immunity factors. Because ROP5 and ROP18 function together to resist innate immune mechanisms, and a significant interaction between them was identified in a two-locus scan, we also assessed the role of ROP18 in the virulence of South American strains. Deletion of ROP18 in South American type 4, 8, and 10 strains resulted in complete attenuation in contrast to a partial loss of virulence seen for ROP18 knockouts in previously described type 1 parasites. These data show that ROP5 and ROP18 are conserved virulence factors in genetically diverse strains from North and South America, suggesting they evolved to resist innate immune defenses in ancestral T. gondii strains, and they have subsequently diversified under positive selection.     

Characterization of a Serine Hydrolase Targeted by Acyl-protein Thioesterase Inhibitors in Toxoplasma gondii

In eukaryotic organisms, cysteine palmitoylation is an important reversible modification that impacts protein targeting, folding, stability, and interactions with partners. Evidence suggests that protein palmitoylation contributes to key biological processes in Apicomplexa with the recent palmitome of the malaria parasite Plasmodium falciparum reporting over 400 substrates that are modified with palmitate by a broad range of protein S-acyl transferases. Dynamic palmitoylation cycles require the action of an acyl-protein thioesterase (APT) that cleaves palmitate from substrates and conveys reversibility to this posttranslational modification. In this work, we identified candidates for APT activity in Toxoplasma gondii. Treatment of parasites with low micromolar concentrations of β-lactone- or triazole urea-based inhibitors that target human APT1 showed varied detrimental effects at multiple steps of the parasite lytic cycle. The use of an activity-based probe in combination with these inhibitors revealed the existence of several serine hydrolases that are targeted by APT1 inhibitors. The active serine hydrolase, TgASH1, identified as the homologue closest to human APT1 and APT2, was characterized further. Biochemical analysis of TgASH1 indicated that this enzyme cleaves substrates with a specificity similar to APTs, and homology modeling points toward an APT-like enzyme. TgASH1 is dispensable for parasite survival, which indicates that the severe effects observed with the β-lactone inhibitors are caused by the inhibition of non-TgASH1 targets. Other ASH candidates for APT activity were functionally characterized, and one of them was found to be resistant to gene disruption due to the potential essential nature of the protein.     

Ciprofloxacin Derivatives Affect Parasite Cell Division and Increase the Survival of Mice Infected with Toxoplasma gondii

Toxoplasmosis, caused by the protozoan Toxoplasma gondii, is a worldwide disease whose clinical manifestations include encephalitis and congenital malformations in newborns. Previously, we described the synthesis of new ethyl-ester derivatives of the antibiotic ciprofloxacin with ~40-fold increased activity against T. gondii in vitro, compared with the original compound. Cipro derivatives are expected to target the parasite’s DNA gyrase complex in the apicoplast. The activity of these compounds in vivo, as well as their mode of action, remained thus far uncharacterized. Here, we examined the activity of the Cipro derivatives in vivo, in a model of acute murine toxoplasmosis. In addition, we investigated the cellular effects T. gondii tachyzoites in vitro, by immunofluorescence and transmission electron microscopy (TEM). When compared with Cipro treatment, 7-day treatments with Cipro derivatives increased mouse survival significantly, with 13–25% of mice surviving for up to 60 days post-infection (vs. complete lethality 10 days post-infection, with Cipro treatment). Light microscopy examination early (6 and 24h) post-infection revealed that 6-h treatments with Cipro derivatives inhibited the initial event of parasite cell division inside host cells, in an irreversible manner. By TEM and immunofluorescence, the main cellular effects observed after treatment with Cipro derivatives and Cipro were cell scission inhibition - with the appearance of ‘tethered’ parasites – malformation of the inner membrane complex, and apicoplast enlargement and missegregation. Interestingly, tethered daughter cells resulting from Cipro derivatives, and also Cipro, treatment did not show MORN1 cap or centrocone localization. The biological activity of Cipro derivatives against C. parvum, an apicomplexan species that lacks the apicoplast, is, approximately, 50 fold lower than that in T. gondii tachyzoites, supporting that these compounds targets the apicoplast. Our results show that Cipro derivatives improved the survival of mice acutely infected with T. gondii and inhibited parasite replication early in the first cycle of infection in vitro, highlighting their therapeutic potential for the treatment of toxoplasmosis.     

Identification of a Post-targeting Step Required for Efficient Cotranslational Translocation of Proteins across the Escherichia coli Inner Membrane

Recent studies have shown that cytoplasmic proteins are exported efficiently in Escherichia coli only if they are attached to signal peptides that are recognized by the signal recognition particle and are thereby targeted to the SecYEG complex cotranslationally. The evidence suggests that the entry of these proteins into the secretory pathway at an early stage of translation is necessary to prevent them from folding into a translocation-incompetent conformation. We found, however, that several glycolytic enzymes attached to signal peptides that are recognized by the signal recognition particle were exported inefficiently. Based on previous studies of post-translational export, we hypothesized that the export block was due to the presence of basic residues at the extreme N terminus of each enzyme. Consistent with our hypothesis, we found that the introduction of negatively charged residues into this segment increased the efficiency of export. Export efficiency was sensitive to the number, position, and sequence context of charged residues. The importance of charge for efficient export was underscored by an in silico analysis that revealed a conserved negative charge bias at the N terminus of the mature region of bacterial presecretory proteins. Our results demonstrate that cotranslational targeting of a protein to the E. coli SecYEG complex does not ensure its export but that export also depends on a subsequent event (most likely the initiation of translocation) that involves sequences both within and just beyond the signal peptide.Since the “signal hypothesis” was proposed over 30 years ago (1), it has become clear that signal sequences are not simply generic hydrophobic peptides that earmark proteins for secretion. In bacteria, the features of a signal peptide determine the mechanism by which a given presecretory protein is targeted to the SecYEG translocation complex in the inner membrane (IM).² Whereas most or all signal peptides are recognized by the signal recognition particle (SRP) in mammalian cells, only a small fraction of Escherichia coli signal peptides are recognized by SRP. These signal peptides are typically extremely hydrophobic (2, 3), but SRP apparently can also recognize slightly less hydrophobic signal peptides that contain a highly basic N terminus (4). SRP recognizes signal peptides as they emerge from translating ribosomes and then targets ribosome-nascent chain complexes to the IM cotranslationally (5). The binding of SRP to its receptor (FtsY), which interacts with the SecYEG complex (6), leads to the release of the nascent chain in the immediate vicinity of the translocation machinery. By targeting nascent polypeptides to the SecYEG complex at an early stage of translation, SRP prevents its substrates from folding into a conformation that is incompatible with translocation through the narrow channel formed by the SecYEG complex (7). Because most signal peptides are not recognized by E. coli SRP, the majority of presecretory proteins are fully synthesized and targeted post-translationally to the IM. These proteins are maintained in a translocation-competent conformation by molecular chaperones such as SecB that keep them unfolded (or loosely folded) (8). Signal peptides themselves also appear to play a role in maintaining translocation competence (9, 10). After mediating the targeting reaction, signal peptides likely play a role in gating open the SecYEG complex to initiate translocation.Interestingly, although signal sequences are the most salient feature of presecretory proteins, they are neither completely necessary nor sufficient to mediate protein export in E. coli (11–13). A version of alkaline phosphatase that lacks a signal peptide is still exported, albeit very inefficiently (11). The export of the leaderless protein, unlike the export of wild-type alkaline phosphatase, is strictly dependent on SecB (11). Conversely, the attachment of signal peptides to cytoplasmic proteins often does not promote their export (14). In light of evidence that folding and export are competing events, these observations led to the proposal that exported proteins tend to fold slowly (or are prevented from folding by chaperones) and therefore remain translocation-competent even without a signal peptide, whereas cytoplasmic proteins fold rapidly into a conformation that is incompatible with export. Recent studies that used thioredoxin as a model protein have validated this hypothesis. Whereas the wild-type protein attached to a typical signal peptide remained trapped in the cytoplasm, four of five slow folding mutants were exported efficiently (15). Furthermore, attachment of a signal peptide that is recognized by SRP to thioredoxin led to efficient export (16). This idea was further confirmed by a report in which various DARPins (designed ankyrin Repeat proteins) were attached to different signal peptides. Most of the DARPins were exported efficiently when they were fused to signal peptides that mediate cotranslational targeting but remained in the cytoplasm when they were attached to signal peptides that are bypassed by SRP (17).Despite these observations, there are several lines of evidence suggesting that export efficiency is not simply dictated by the ability of a protein to reach the SecYEG complex before folding into a translocation-incompetent conformation. For reasons that are unclear, some DARPins are secreted inefficiently even when they are routed into the SRP pathway (17). In addition, numerous reports have indicated that the amino acid composition of the segment of post-translationally targeted presecretory proteins that lies just beyond the signal peptide cleavage site has a dramatic effect on export efficiency. Statistical analysis has shown that the first ∼5–15 residues of the mature region of most presecretory proteins produced by Gram-negative bacteria is neutral or has a net negative charge (18). Consistent with the observed sequence bias, the presence of multiple basic residues at the N terminus of the mature region often leads to accumulation of the secretory precursor, whereas conversion of the basic residues to acidic residues restores export (19–22). Because different combinations of proteins and signal peptides were used in these studies, the exact number and location of charged residues that impinge on the efficiency of export is unclear. In any case, the effect of the net charge in the region distal to the signal peptide on protein export has never been explained. Although basic residues might conceivably promote premature folding of presecretory proteins or block the cleavage of signal peptides by leader peptidase, it is also possible that they inhibit an uncharacterized post-targeting event. Even if effects on signal peptide cleavage could have been ruled out in the aforementioned studies, however, it would not have been possible to distinguish between effects on protein folding and effects on a hypothetical post-targeting step because only proteins that are targeted post-translationally were monitored.To gain further insight into the factors that govern the efficiency of protein export, we sought an explanation for the observation that the cotranslational targeting of at least some cytoplasmic proteins is insufficient to guarantee their translocation across the IM. We found that the export of several different endogenous E. coli cytoplasmic proteins required not only the attachment of a signal peptide that is recognized by SRP but also a net negative charge just past the signal peptide cleavage site. Taken together with previous results, our data show that the charge of the segment just beyond the signal peptide influences export efficiency irrespective of the mechanism by which a protein is targeted to the IM. Because proteins that are targeted cotranslationally reach the IM before they have a chance to fold, our results imply the existence of a post-targeting step (most likely the initiation of translocation) that is facilitated by acidic residues distal to the signal peptide and inhibited or delayed by basic residues. These results help to resolve a long-standing puzzle about the influence of the mature region of presecretory proteins on protein export and have significant implications for optimizing the export of cytosolic and heterologous proteins in E. coli.